Bottom Line:
Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition.No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain.We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

Affiliation: Department of Neurology, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

ABSTRACTApolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

Mentions:
Studies have shown that ApoE isoforms have different kinetic properties in peripheral plasma [13]–[15]. In order to confirm that our ApoE isoform-specific LC/MS method yielded similar findings [29], plasma ApoE samples were analyzed from individuals labeled with 13C6-leu. CSF and blood were collected for 48 hours [30], [31]. ApoE labeling patterns obtained from an individual with each ApoE genotype are shown in Figure 1. The plasma ApoE TTR (tracer to tracee ratio) time course was characterized by a rapid rise during tracer infusion over 9 hours, followed by clearance of labeled ApoE. The TTR maximum at 10 h was 20% for ApoE4, 10% for ApoE3 and 4.5% for ApoE2 (Fig. 1) demonstrating that the isoforms have different turnover rates. The peripheral ApoE compartmental model (Fig. 2A) provided a strong fit to all sets of ApoE data (Fig. 2B–D). Kinetic parameters [fractional synthesis rate (FSR), monoexponential slope fractional clearance rate (FCR), and compartmental model fractional turnover rate (FTR)] of plasma ApoE are summarized in Table 1. Differences in ApoE isoform kinetics were found when each individual isoform was analyzed both between homozygous subjects and within heterozygous subjects. For example, ApoE4 protein turnover rates in homozygous subjects were approximately twice as fast the ApoE3 turnover rates in homozygous subjects (Fig. 1A–B, Table 1). Gregg et al. also observed in homozygotes that ApoE4 was catabolized 2-fold faster than ApoE3 [15]. Within the same subject, ApoE4 kinetic rates were double that of ApoE3 kinetic rates (Fig. 1C, Table 1). Furthermore, the LAVYQAGAR peptide, common to ApoE3 and ApoE4 proteins, exhibited kinetics that were intermediate between the plasma ApoE3 and ApoE4 peptides within ApoE3/4 heterozygotes (Table 1). ApoE2 had the slowest turnover rate. The plasma ApoE4 turnover rate was approximately 4-fold faster than plasma ApoE2 within ApoE2/4 heterozygotes (Table 1), which is similar to the 3-fold difference previously reported by Ikewaki et al. [13]. Therefore, consistent with previously reports [13]–[15], the plasma ApoE isoforms have different turnover rates, confirmed here using our isoform-specific LC/MS technique. ApoE protein levels in plasma also trended towards being lower in ApoE4 carriers (Table S1).

Mentions:
Studies have shown that ApoE isoforms have different kinetic properties in peripheral plasma [13]–[15]. In order to confirm that our ApoE isoform-specific LC/MS method yielded similar findings [29], plasma ApoE samples were analyzed from individuals labeled with 13C6-leu. CSF and blood were collected for 48 hours [30], [31]. ApoE labeling patterns obtained from an individual with each ApoE genotype are shown in Figure 1. The plasma ApoE TTR (tracer to tracee ratio) time course was characterized by a rapid rise during tracer infusion over 9 hours, followed by clearance of labeled ApoE. The TTR maximum at 10 h was 20% for ApoE4, 10% for ApoE3 and 4.5% for ApoE2 (Fig. 1) demonstrating that the isoforms have different turnover rates. The peripheral ApoE compartmental model (Fig. 2A) provided a strong fit to all sets of ApoE data (Fig. 2B–D). Kinetic parameters [fractional synthesis rate (FSR), monoexponential slope fractional clearance rate (FCR), and compartmental model fractional turnover rate (FTR)] of plasma ApoE are summarized in Table 1. Differences in ApoE isoform kinetics were found when each individual isoform was analyzed both between homozygous subjects and within heterozygous subjects. For example, ApoE4 protein turnover rates in homozygous subjects were approximately twice as fast the ApoE3 turnover rates in homozygous subjects (Fig. 1A–B, Table 1). Gregg et al. also observed in homozygotes that ApoE4 was catabolized 2-fold faster than ApoE3 [15]. Within the same subject, ApoE4 kinetic rates were double that of ApoE3 kinetic rates (Fig. 1C, Table 1). Furthermore, the LAVYQAGAR peptide, common to ApoE3 and ApoE4 proteins, exhibited kinetics that were intermediate between the plasma ApoE3 and ApoE4 peptides within ApoE3/4 heterozygotes (Table 1). ApoE2 had the slowest turnover rate. The plasma ApoE4 turnover rate was approximately 4-fold faster than plasma ApoE2 within ApoE2/4 heterozygotes (Table 1), which is similar to the 3-fold difference previously reported by Ikewaki et al. [13]. Therefore, consistent with previously reports [13]–[15], the plasma ApoE isoforms have different turnover rates, confirmed here using our isoform-specific LC/MS technique. ApoE protein levels in plasma also trended towards being lower in ApoE4 carriers (Table S1).

Bottom Line:
Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition.No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain.We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.

Affiliation:
Department of Neurology, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

ABSTRACTApolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer's disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer's disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis.